Germination is one of the most important “decisions” in the life-history of a plant, as it sets in motion the growth of the seedling. Therefore it is not surprising that seed dormancy is a complex trait, influenced by a myriad of genetic and environmental factors that interact to maximize the long-term chance of survival of the seed (Koornneef et al., 2002). Many of these factors are mediated by hormones, with gibberellic acid (GA), ethylene and brassinosteriods all known to promote germination, and abscisic acid (ABA) known to promote dormancy (Koornneef et al., 2002).
During seed development, ABA content increases and regulates many key processes including the imposition and maintenance of dormancy (Bewley, 1997). This is illustrated by ABA-deficient mutants of maize that display vivipary (McCarty, 1995) and by ABA-deficient mutants of Arabidopsis that can germinate in the absence of GA (Koornneef et al., 1982). Upon imbibition, this high level of ABA must be reduced in order for the seeds to germinate, and recent studies have shown that this occurs when dormancy is broken by after-ripening, stratification, dark or treatment with smoke water (for review see Gubler et al., 2005). For example, in after-ripened non-dormant (ND) Arabidopsis seeds, upon imbibition ABA levels decline rapidly after which germination proceeds (Ali-Rachedi et al., 2004). In contrast, although the ABA level also declines in imbibed dormant (D) seed, it does so to a lesser extent and only transiently, with ABA content again increasing to levels similar to that observed in the non-imbibed seeds (Ali-Rachedi et al., 2004).
Physiological processes mediated by ABA are usually correlated with fluctuating endogenous levels of the hormone. These levels may be regulated through the balance of biosynthesis and catabolism, with biosynthesis dominating when levels are increasing and catabolism dominating when levels are decreasing. In plants, ABA is synthesized indirectly from carotenoids (Seo and Koshiba, 2002), with the first committed step being catalyzed by the enzyme, 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, an ABA precursor (Schwartz et al., 1997). A direct correlation between NCED mRNA levels, NCED protein and ABA levels in water-stressed tissues suggest that this step plays a strong regulatory role in many plant species (Qin and Zeevaart, 1999). Additional evidence comes from several instances of over-expression of this enzyme in transgenic plants which leads to higher ABA levels that direct physiological processes (Iuchi et al., 2001) including increasing seed dormancy (Thompson et al., 2000; Qin and Zeevaart, 2002).
ABA levels may not be controlled solely by NCED, as overexpression of a gene encoding zeaxanthin epoxidase (ZEP) in transgenic tobacco resulted in increased ABA levels in mature seed and greater seed dormancy, although the effect of ZEP overexpression was limited (Frey et al., 1999). Furthermore, treatment with the ABA biosynthesis inhibitor fluridone (Grappin et al., 2000) can reduce seed dormancy, indicating that ABA biosynthesis is active in the imbibing seed. Together these experiments indicate that increased ABA biosynthesis can lead to greater seed dormancy.
Several alternative catabolic pathways exist for the inactivation of ABA (Cutler and Krochko, 1999; Zhou et al., 2004). One pathway is the hydroxylation of ABA at the 8′-position to produce 8′-hydroxy ABA, which spontaneously isomerizes to phaseic acid (PA) (Cutler and Krochko, 1999). PA may be further catabolised by a reductase to dihydrophaseic acid (FIG. 1).
In both barley and Arabidopsis, the decrease in ABA during seed imbibition is associated with increases in PA, consistent with this catabolism pathway (Jacobsen et al., 2002; Kushiro et al., 2004). The first reaction is catalysed by a cytochrome P450 monooxygenase known as ABA 8′-hydroxylase (ABA8′OH, Krochko et al., 1998). The AtCYP707A1-AtCYP707A4 gene subfamily of Arabidopsis has recently been shown to encode ABA8′OH activity via heterologous expression in yeast (Kushiro et al., 2004) or insect cells (Saito et al., 2004). Expression studies in Arabidopsis showed that AtCYP707A2 was associated with the rapid decline of ABA in germinating seeds (Kushiro et al., 2004). Furthermore, seeds of cyp707a2 mutants accumulated more than six-fold greater ABA content than wild type seeds and consequently were hyperdormant (Kushiro et al., 2004).
Dormancy is an important agricultural trait, with too little dormancy leading to pre-harvest sprouting in cereals (early germination of grains in the head in moist conditions), or too much dormancy causing inability to germinate, delayed or non-uniform germination, all of which would give rise to poor establishment of crops in the field and poor grain performance in processes such as malting of barley. Two proteins have been identified from rice which are involved in inactivation of ABA (WO2004/113527). However, there is a need for cereals with modified dormancy, in particular of barley and wheat.